Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To study the mechanism of basal transcription by RNA polymerase II, a cDNA encoding the Drosophila homologue of the small subunit of TFIIF (also referred to as TFIIF30, RAP30, factor 5b, and gamma) was isolated. The Drosophila TFIIF30 gene is located at region 86C on the right arm of the third chromosome. The protein encoded by the cDNA, termed dTFIIF30, was synthesized in Escherichia coli and purified to greater than 95% homogeneity. In reconstituted transcription reactions with purified basal factors, the specific activity of dTFIIF30 was identical to that of its human homologue. Moreover, a carboxyl-terminal fragment, designated dF30(119-276), which contains the carboxyl-terminal 158 amino acid residues of dTFIIF30, was found to possess approximately 50% of the transcriptional activity as full-length dTFIIF30. The interaction of dTFIIF30 with the large subunit of TFIIF (also referred to as TFIIF74, RAP74, factor 5a, and beta) was investigated by glycerol gradient sedimentation analyses. In these experiments, dTFIIF30, but not dF30(119-276), assembled into a stable heteromeric complex with TFIIF74. These results, combined with those of previous work on TFIIF, support a model for TFIIF30 function in which the carboxylterminal region constitutes a functional domain that can interact with RNA polymerase II to mediate basal transcription, whereas the amino terminus comprises a domain that interacts with TFIIF74.
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PMID:Structure and function of the small subunit of TFIIF (RAP30) from Drosophila melanogaster. 789 Jul 67

Mammalian transcription factor IIF (TFIIF) is a heterodimer composed of approximately 30-kDa (RAP30) and approximately 70-kDa (RAP74) subunits. TFIIF has been shown to bind RNA polymerase II and control the activity of the enzyme in both the initiation and elongation stages of transcription. Although previous studies have established a role for RAP30 in assembly of the preinitiation complex and in transcription initiation, information on the function of RAP74 in these processes has been lacking. Using a highly purified transcription system and assays that permit sensitive measurement of the contributions of both RAP30 and RAP74 to TFIIF function, we have investigated the roles of these TFIIF subunits in transcription initiation and elongation. Results of template competition experiments indicate that both RAP30 and RAP74 contribute to the formation of stable preinitiation intermediates containing RNA polymerase II. Investigation of the role of TFIIF in transcription initiation indicates that both RAP30 and RAP74 function in synthesis of the first few phosphodiester bonds of nascent transcripts and in formation of Sarkosyl-resistant pre-initiation intermediates. Finally, kinetic experiments indicate that both RAP30 and RAP74 function in TFIIF-mediated stimulation of the rate of RNA chain elongation by RNA polymerase II.
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PMID:Roles for both the RAP30 and RAP74 subunits of transcription factor IIF in transcription initiation and elongation by RNA polymerase II. 792 73

The human general transcription factor IIF (TFIIF) is required for an accurate transcription initiation by RNA polymerase II and shares some analogous features with the sigma subunit of bacterial RNA polymerase. As an attempt to analyze the function of TFIIF, we examined its effect on bacterial transcription in vitro. TFIIF significantly enhanced the initiation of transcription by the bacterial RNA polymerase while other general transcription factors, TATA-binding protein, TFIIB, and TFIIE, did not. The enhancement of the bacterial transcription was ascribed to the 74 kDa subunit of TFIIF (RAP74). RAP74 had an activity of enhancing the binding of the bacterial RNA polymerase to the promoter. The enhancing activity of RAP74 depended on a low molar ratio of the RNA polymerase to the template DNA. The action of RAP74 in the bacterial transcription may be related to a possible regulatory role of RAP74 in the eukaryotic transcription initiation.
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PMID:Enhancement of bacterial transcription initiation in vitro by the 74 kDa subunit of human general transcription factor IIF (RAP74). 794 16

The transcription initiation factor, TFIIF, is essential not only for the initiation of transcription but also for efficient elongation of mRNA synthesis by mammalian RNA polymerase II and is extensively phosphorylated in vivo. The possible regulation of TFIIF activity by protein phosphorylation was investigated by comparing the biochemical properties of alkaline phosphatase-treated HeLa TFIIF with those of native or bacterially expressed factor. Alkaline phosphatase treatment decreased the size of the large subunit (RAP74) of TFIIF to that of the recombinant protein but did not change the size of the small subunit (RAP30). Both the transcription initiation and elongation stimulating activities of the alkaline phosphatase-treated TFIIF decreased to 15-20% of the native form under conditions in which the amount of TFIIF was rate-limiting for transcription. Furthermore, phosphatase-treated TFIIF assembled the DBPolF complex and bound to RNA polymerase II less efficiently than the native protein. When hybrid TFIIFs were reconstituted using native or recombinant subunits, a native form of RAP74 stimulated both transcription and DBPolF complex formation activity regardless of whether native or recombinant RAP30 was used. We propose that TFIIF activity is regulated by protein phosphorylation, particularly of the RAP74 subunit. The functional role of RAP74 in assembling the preinitiation complex and modulating TFIIF activity is discussed.
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PMID:Regulation of the human general transcription initiation factor TFIIF by phosphorylation. 796 96

General transcription factors are required for accurate initiation of transcription by RNA polymerase II. Human cDNAs encoding subunits of these factors have been cloned and sequenced. Using fluorescence in situ hybridization (FISH), we show here that the genes encoding the TATA-box binding protein (TBP), TFIIB, TFIIE alpha, TFIIE beta, RAP30, RAP74 and the 62 kDa subunit, of TFIIH are located at the human chromosomal bands 6q26-27, 1p21-22, 3q21-24, 8p12, 13q14, 19p13.3 and 11p14-15.1, respectively. This dispersed localization of a group of functionally related gene provides insights into the molecular mechanism of human genome evolution and their possible involvement in human diseases.
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PMID:Genes encoding general initiation factors for RNA polymerase II transcription are dispersed in the human genome. 816 52

The general transcription factor TFIIE, together with other general transcription factors, is essential for transcription initiation by RNA polymerase II. TFIIE stimulates the TFIIH-dependent kinase activity that phosphorylates the carboxy-terminal domain of the largest subunit of RNA polymerase II, and possesses a helicase activity. Here we show that human TFIIH has DNA-dependent ATPase activity and we characterize the stimulatory effect of TFIIE on both the ATPase and kinase activities. We demonstrate that extensive phosphorylation of RNA polymerase II occurs in a TFIIE-dependent manner in both the absence and presence of DNA but, in the latter case, only at a late stage of preinitiation complex assembly. We also show that TFIIH specifically phosphorylates three general transcription factors, human TFIID tau (TBP), TFIIE-alpha and TFIIF-alpha (RAP74).
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PMID:Regulation of TFIIH ATPase and kinase activities by TFIIE during active initiation complex formation. 816 91

Factor 5 is a Drosophila RNA polymerase II initiation factor that also affects the elongation phase of transcription. We have used a cDNA encoding the large subunit of factor 5 (F5a) to produce recombinant F5a (rF5a). Antibodies directed against peptides deduced from the sequence of the F5a cDNA recognized rF5a and the large subunit of factor 5 purified from Kc cells. A chimeric human/fly factor composed of the small subunit of human TFIIF (RAP30) and rF5a stimulated elongation by Drosophila RNA polymerase II when assayed using a dC-tailed template. In addition, the chimeric human/fly factor functioned during initiation in either the Drosophila or human system. Therefore, the structure of the large subunit of TFIIF is sufficiently conserved from human to fly to allow functional interaction with both the small subunit of TFIIF and RNA polymerase II from either species. Analysis of deletion mutants of F5a indicated that almost all of the protein was required for initiation while only the NH2-terminal region was required for stimulating transcriptional elongation. A comparison of our results with those obtained with RAP74 suggest that the carboxyl terminal region of the protein may be involved in interactions with RNA polymerase II or other factors during initiation.
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PMID:Functional analysis of Drosophila factor 5 (TFIIF), a general transcription factor. 817 88

RNA polymerase II-associating proteins (RAP30 and RAP74) are subunits of the transcription factor called variously RAP30/74, TFIIF, beta gamma, and FC. This factor is required for accurate transcription by RNA polymerase II, in addition to other basal transcription factors. Using recombinant human RAP30 and RAP74, the functions of these subunits have been tested separately during the initiation and elongation phases of transcription. RAP30 is required to form a Sarkosyl-resistant complex at 0.25% Sarkosyl, so RAP30 is required for initiation. RAP74, however, stimulates transcription when added after Sarkosyl, indicating that RAP74 is dispensable for initiation. The same result is obtained using a pulse-chase protocol in which accurately initiated RNA is labeled during a short pulse, followed by a chase with excess unlabeled nucleoside triphosphates. RAP30 is required in order to label the transcript during the pulse, but RAP74 is not. RAP74 must be added during the chase, however, in order to obtain a short runoff transcript. The following conclusions can be drawn from these experiments: 1) RAP30 is an initiation factor; 2) RAP74 is not required for ATP hydrolysis in initiation, which precedes phosphodiester bond formation; 3) RAP74 is not required for template strand separation; 4) RAP74 is not required to initiate phosphodiester bond formation; and 5) RAP74 is required for very early elongation.
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PMID:RAP30/74 (transcription factor IIF) is required for promoter escape by RNA polymerase II. 837 3

RAP30 and RAP74 are subunits of RAP30/74 (TFIIF), a general initiation and elongation factor for transcription by RNA polymerase II. Complementary DNA (cDNA) clones have previously been reported encoding human RAP30 and RAP74. Here we report expression of these cDNAs using a T7 RNA polymerase system in Escherichia coli. Production of human RAP30 was very efficient using the expression vector pET11d. RAP30 accumulated within inclusion bodies and was solubilized using guanidine hydrochloride. After removal of the denaturant, RAP30 was soluble and active in accurate transcription. Approximately 44 mg of highly purified and soluble RAP30 was obtained from a 1-liter culture of cells. Production of RAP74 was more problematic, because a mixture of full length RAP74 and RAP74 fragments was produced in E. coli. Most RAP74 fragments were shortened by deletion of the COOH-terminus of the protein and probably resulted from premature translation termination. RAP74 was most successfully produced using a pET23d construct, in which the RAP74 peptide was fused to a short polyhistidine stretch at its COOH-terminus. Addition of the polyhistidine sequence allowed purification using a Ni2+ affinity resin. Full length RAP74 carrying this polyhistidine extension was purified in a single step by Ni2+ affinity chromatography in 4 M urea; the yield of RAP74 was approximately 3 mg from a 1-liter culture of cells. RAP74 derivatized with a polyhistidine stretch at its NH2-terminus, on the other hand, remained contaminated with RAP74 fragments after Ni2+ affinity chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Production of human RAP30 and RAP74 in bacterial cells. 839 Aug 79

The structural and functional domains of a general transcription initiation factor, TFIIF (RAP30/74, FC), have been investigated using various deletion mutants of each subunit, both in vivo and in vitro. An in vivo assay showed that the N-terminal sequence containing residues of 1-110 of RAP30 that is located close to a sigma homology region interacts with a minimum sequence of residues 62-171 of RAP74 to form a heteromeric interaction. Reconstitution of in vitro transcription activity by deletion mutants of RAP74 clearly indicated that both N-terminal residues 73-205 and C-terminal residues 356-517 are essential for full activity, the former interacting with RAP30, thus complexing with RNA polymerase II. From these data, the functional significance of domain structure of TFIIF is discussed in terms of its sigma homology sequences and complex formation with RNA polymerase II in the initiation and elongation of transcription.
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PMID:Domain structure of a human general transcription initiation factor, TFIIF. 844 35


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